www.nature.com/scientificreports OPEN Hidden mycota of pine needles: Molecular signatures from PCR- DGGE and Ribosomal DNA Received: 24 April 2018 Accepted: 9 November 2018 phylogenetic characterization of Published: xx xx xxxx novel phylotypes Rajesh Jeewon1, Quin S. Y. Yeung2, Dhanushka N. Wannasinghe3,6, Sillma Rampadarath4, Daneshwar Puchooa4, Hong-Kai Wang5 & Kevin D. Hyde3 Previous studies for enumerating fungal communities on pine needles relied entirely on phenotypic diversity (microscopy) or identifcation based on DNA sequence data from those taxa recovered via cultural studies. To bypass limitations of the culturing methods and provide a more realistic diversity estimate, we employed and assessed a PCR-DGGE based method coupled with rDNA phylogenetic sequence analyses to characterize fungal taxa associated with pine needles. Fresh (living) and decayed needles from three hosts of the Pinaceae (Keteleeria fortunei, Pinus elliottii and P. massoniana) were examined. Morphological studies reveal that the most abundant species associated with decayed needles were Cladosporium cladosporioides and an unidentifed Trichoderma species followed by Gliocephalotrichum sp., Gliocladium sp., Lophodermium pinastri, Paecilomyces varioti, Phaeostalagmus cyclosporus and a Phoma sp, which are commonly occurring fungi. Community genomic data from freshly collected and decayed pine needles recovered 40 operational taxonomic units, which appear to be mostly undetected members of the natural fungal consortium. Sequence analyses revealed a number of phylotypes or “species” that were not recovered using traditional morphological and cultural approaches previously used. Phylogenetic data from partial 18S rDNA sequence data reveal that most phylotypes represent potential novel phylogenetic fungal lineages with afnities to the Dothideomycetes, Leotiomycetes, Lecanoromycetes and Sordariomycetes and were not identical to previously known endophytes or saprobes. Although the major ecological roles of these phylotypes in pine needles are still enigmatic, this study provides new insights in hidden fungal diversity that mycologists are possibly ignoring given the discrepancies associated with available methods. To what extent do previously recovered identifed species (either as saprobes or endophytes) from morphological or culturing studies act as pioneer decomposers or constitute an integral part of endophytic community warrants further investigation. Fungi, especially from pines, are important contributors to primary productivity and nutrient cycling. Tey are mostly isolated as endophytes, pathogens or saprobes and are critical components in plant ecosystems1–3. Several studies have focused on the characterization of fungi colonising pine needles from ecological and patho- logical perspectives (e.g.4–10). Te pioneering work on mycota of pine litter by Kendrick and Burges4 prompted other researchers to focus on this host, which is an important component of many forests. Traditional methods 1Department of Health Sciences, Faculty of Science, University of Mauritius, Reduit, Mauritius. 2School of Biological Sciences, Faculty of Science, University of Hong Kong, Pokfulam Rd, Hong Kong, SAR, China. 3Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai, 57100, Thailand. 4Faculty of Agriculture, University of Mauritius, Réduit, Mauritius. 5Biotechnology Institute, Zhejiang University, 310029, Zhejiang, P. R. China. 6Key Laboratory for Plant Biodiversity and Biogeography of East Asia (KLPB), Kunming Institute of Botany, Chinese Academy of Science, Kunming, 650201, Yunnan, China. Correspondence and requests for materials should be addressed to R.J. (email: [email protected]) SCIENTIFIC REPORTS | (2018) 8:18053 | DOI:10.1038/s41598-018-36573-z 1 www.nature.com/scientificreports/ documenting fungal diversity, however, rely mostly on taxonomic identifcation of species based on spore mor- phology and DNA sequence data of endophytes from cultural dependent methods. Tis has a major drawback given that not all fungi grow readily in highly selective medium and sporulation is dependent on species, host, seasonality, growth conditions, and other environmental factors which can largely underestimate diversity. In many cases, fungi exist predominantly as vegetative hyphae, which make microscopic examination dif- cult3,7. In addition, the use of artifcial media does not refect natural conditions and only those microorganisms that thrive well under these conditions will grow. It is thought that only a small fraction (0.1 to 10%) of micro- organisms existing in the nature can be cultured artifcially11. Te use of previous classical methods to enumer- ate fungal taxa or characterise fungal communities from pine needles has therefore underestimated diversity. Generally, most studies were limited to temperate regions and have consistently isolated only a few dominant fungi, the most common of which are Lophodermium pinastri and L. piceae12. Recently, several studies based on DNA sequence analyses, especially those targeting endophytes, have also revealed diferent groups of fungal species not recovered via traditional methods (e.g.8–10). To circumvent the challenges associated with spore production and cultivation, a number of molecular based approaches have been widely used (as detailed in Anderson et al.13). Among these, PCR-DGGE has proven to be a powerful technique to detect and characterize fungal communities in soil samples, plant material (roots and leaves), rocks, water, alga and glass13–17. Studies have revealed that dominant microorganisms isolated from envi- ronmental samples using traditional and cultivation-dependent methodology are genetically diferent from those identifed via DGGE analysis of rDNA gene (e.g.18). Diversity of freshwater fungi in decaying leaves of white birch (Betula papayrifera), several maple species (Acer rubrum, A. saccharum, and A. spicatum) and white spruce (Picea glauca) has been reported using a traditional and a molecular approach19. Tey found that species richness esti- mates based on DGGE are higher than those from T-RFLP analysis and much higher than those based on spore identifcation. Subsequent DGGE analyses of aquatic hyphomycetes have also revealed that presence or absence of fungal taxa and biomass from community DNA is regulated primarily by season and type of substrates20. Based on sequence analyses of DGGE bands, Vainio & Hantula15 successfully detected a higher fungal diversity from envi- ronmental samples of Picea abies (Norway spruce stumps) than when using culture techniques. Cullings et al.21 used DGGE to evaluate changes in soil communities in response to the efects of artifcial defoliation of pines in a pine-spruce forest. From a pathological perspective, DGGE has also been valuable in profling fungal taxa that are associated with healthy and infected black spruce seedlings (Picea marina)22. Our previous studies on pine needles from Keteleeria fortunei, Pinus elliottii and Pinus massoniana (Family: Pinaceae) in Hong Kong revealed a few taxa new to science23,24 and a large number of unidentifed taxa, especially those that exist predominantly in their asexual stages and sterile hyphae. To the best of our knowledge, there is no known studies on pine needles of these 3 members of the Pinaceae that have analysed the fungal species (espe- cially those colonizing internal tissues) based on PCR-DGGE followed by 18S rDNA sequence analyses. Mahajan et al.9 have attempted to characterize microbial communities from Pinus roxburghii of the Himalayan subtropical region (India) based on PCR-DGGE using ITS-GC clamped primers but all PCR amplifcations failed and no fungal taxa could be sequenced. Aneja et al.25 used DGGE to characterize bacteria and fungi but their focus was detection of potential saprobic fungi that are potential decomposers in spruce planted in vitro. In this study we attempt to recover fungal species found in pine needles using a polyphasic approach (direct morphological com- parison, cultural based methods and PCR-DGGE analyses of 18S rDNA coupled with phylogenetic analyses). Te objectives of this study were to (1) profle the main fungal species associated with diferent decay stages and (2) fnd out whether fungal species from pine needles are more diverse than expected and, (3) whether “taxa” recovered from the molecular approach are related to previously identifed endophytes or whether they constitute unknown phylogenetic lineages. Results Morphological-based methodology. A total of 39 taxa were identifed from the three hosts and 2 needle types (Table 1). A large number of anamorphic (asexual) fungi (77%) were recovered, while the average occur- rence of sexual morph ascomycetes was quite low (6.25%). Te most abundant taxa were Cladosporium clad- osporioides, Gliocephalotrichum sp., Gliocladium sp., Paecilomyces varioti, Phaeostalagmus cyclosporus, Phoma sp., and a Trichoderma sp. Te percentage of non-sporulating taxa was 14.58%. A proportion of fungi (14.5%) existed as cultures that could not be identifed as no sporulating structures were observed. Tese were termed as “Mycelia sterilia” (sensu3,26) and have the highest frequency of occurrence. Lophodermium pinastri has the highest fre- quency of occurrence among the three ascomycetes present. Anamorphic fungi were dominant on both, L and F needle types (with 100% dominance on Keteleeria fortunei (L-type needles), 94.74% on K. fortunei (F-type needles), 96.77% on Pinus elliottii and 93.75% on P. massoniana). Te highest species diversity